26 March 2014

Researchers from Lawrence Livermore National Laboratory in conjunction with the Joint BioEnergy Institute (JBEI) have engineered tolerance to ionic liquids (ILs)—used for biomass pretreatment, but generally toxic to bacteria—into biofuel-producing bacteria.

The results, reported in an open access paper in Nature Communications are likely to eliminate a bottleneck in JBEI’s biofuels production strategy, which relies on ionic liquid pretreatment of cellulosic biomass. The research also demonstrates how the adverse effects of ionic liquids can be turned into an advantage, by inhibiting the growth of other bacteria.

The inherent recalcitrance of biomass requires an initial pretreatment step to render polysaccharides free from lignin for subsequent enzymatic or chemical hydrolysis to fermentable sugars. To solubilize lignocellulosic biomass, certain hydrophilic ILs are highly effective and environmentally friendly pretreatment agents that generate relatively low amounts of biomass-derived inhibitors compared with other conventional pretreatment methods. A major disadvantage of the commonly used imidazolium ILs is their intrinsic microbial toxicity, which impairs growth of typical biofuel-producing hosts such as Escherichia coli and Saccharomyces cerevisiae, hence preventing efficient biofuel production. In addition, the inhibition of biofuel synthetic enzymes by these ILs can severely reduce the yield of the final product.

It was recently demonstrated that an engineered E. coli strain is able to convert IL-pretreated biomass into biofuels in laboratory-scale experiments. However, the extensive washing required for complete IL removal is not feasible in large-scale, industrial applications. An ideal and more sustainable process should balance the costs of removing IL with fermentation performance. A novel way to achieve this would use biofuel-producing microbes that can tolerate residual levels (for example, 0.2–5% wt/vol) of ILs.

—Ruegg et al.

At JBEI, researchers have previously engineered strains of E. coli bacteria to digest the cellulosic biomass of switchgrass, a perennial grass that thrives on land not suitable for food crops, and convert its sugars into biofuels and chemicals. However, the ionic liquids used to make the switchgrass digestible proved to be too toxic for the E. coli and had to be completely removed through several washings prior to fermentation.

The team identified an IL-resistance mechanism consisting of two adjacent genes from Enterobacter lignolyticus, a rain forest soil bacterium that is tolerant to an imidazolium-based IL. These genes retained their full functionality when transferred to an E. coli biofuel host. The genetic module provided the tolerance needed for the E. coli to grow well in the presence of toxic concentrations of ionic liquids.

Bisabolane is an advanced biofuel candidate with combustion properties comparable to diesel. The dehydrogenated precursor, bisabolene, is produced from acetyl-coenzyme A (CoA) in E. coli with a two-plasmid system: one plasmid contains eight heterologous mevalonate pathway genes, and the other has the plant-derived bisabolene synthase gene. We assembled these genes onto a single plasmid optimized for bisabolene production, then introduced a second plasmid containing the eilAR cassette to create a prototype IL-tolerant biofuel production system.

—Ruegg et al.

They tested cell growth and bisabolene production by this engineered E. coli strain in a medium containing a range of IL concentrations from 0 to 270 mM, representing the residual IL remaining with the hydrolysate after pretreatment and saccharification.

They found that the new genes confer to E. coli the ability to grow in the presence of normally toxic levels of an ionic liquid.

Gray bars represent E. coli with the plasmid containing the bisabolene pathway but without the IL-resistant plasmid (eilAR cassette); blue bars represent E. coli with the IL-resistant capability. (a) Time required for each culture to reach OD600nm=1, the standard cell density used for inducing the bisabolene pathway. (b) Bisabolene production measured 6 days after induction of cultures grown in three IL concentrations, as indicated with columns and error bars representing the means and standard deviation of technical triplicate measurements. Values were normalized to cell density to illustrate productivity of the bacteria. X indicates that no growth or production was observed. Ruegg et al. Click to enlarge.

JBEI researchers used an approach devised by lead author and Basel University graduate student and LLNL guest researcher Thomas Ruegg to \pinpoint rapidly the genes responsible for ionic liquid resistance in the genomic DNA of Enterobacter lignolyticus.

This genetic module encodes both a membrane transporter and its transcriptional regulator. While a pump exports ionic liquids, the substrate-inducible regulator maintains the appropriate level of this pump so that the microbe can grow normally either in the presence or absence of ionic liquid.